Chimeric antigen receptor specific for interleukin-23 receptor
A chimeric antigen receptor, IL-23 technology, applied in the field of immunotherapy, can solve problems such as inability to obtain
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no. 1 approach
[0272] According to a first embodiment, the CAR of the invention comprises an extracellular IL-23R binding domain, optionally an extracellular hinge domain, a transmembrane domain and an intracellular signaling domain.
[0273] In one embodiment, the CAR of the present invention comprises an IL-23R binding domain; a CD8 transmembrane domain (preferably SEQ ID NO: 21); and a CD3-ζ first signaling domain (preferably SEQ ID NO: 25 , 26, 61 or 62).
[0274] In one embodiment, the CAR of the present invention comprises an IL-23R binding domain; a CD28 transmembrane domain (preferably SEQ ID NO: 23); and a CD3-ζ first signaling domain (preferably SEQ ID NO: 25 , 26, 61 or 62).
[0275]In another embodiment, the CAR of the present invention comprises an IL-23R binding domain; a hinge domain of CD8 (preferably SEQ ID NO: 13); a transmembrane domain of CD8 (preferably SEQ ID NO: 21); and CD3 - zeta first signaling domain (preferably SEQ ID NO: 25, 26, 61 or 62).
[0276] In one embo...
Embodiment 1
[0597] Example 1: Anti-IL23RCAR Human Treg
[0598] Materials and Methods
[0599] Cells and Reagents
[0600] HEK293T cells (LentiX, Ozyme, France) were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS).
[0601] Isolation of FoxP3 Treg
[0602] Using EasySepTM human CD4 purchased from StemCell + CD127 低 CD25 + Regulatory T Cell Isolation Kit, freshly isolated CD4 from buffy coat + CD25 + CD127 低 Treg cells. After isolation, purity was assessed by FoxP3 staining. The isolated Treg cells were divided into 5 × 10 per well 5 Cells were seeded in X-VIVO 15 medium (Lonza) in 24-well plates (Costar) and coated with anti-CD3 / anti-CD28 microbeads (Invitrogen, Carlsbad, CA) at a bead to cell ratio of 1:1. activation. Treg cells were activated while receiving rapamycin (100 ng / ml). IL-2 (1000 units / ml, Miltenyi) was added on days 2, 5, 7 and 9.
[0603] CAR construct
[0604]The anti-IL23R CAR construct comprises a human CD8 leader sequence (aa1-22...
Embodiment 2
[0616] Example 2: Anti-IL23R CAR mouse Treg
[0617] Materials and Methods
[0618] Isolation of FoxP3 Treg
[0619] CD4 was freshly isolated from the spleen of C57B16 mice using Life Technologies' Regulatory T Cell Isolation Kit + CD25 + Mouse Treg cells. After isolation, purity was assessed by FoxP3 staining. The isolated Treg cells were divided into 5 × 10 per well 5 10 cells were seeded in RPMI 10% SVF in 24-well plates and activated with anti-CD3 / anti-CD28 coated microbeads (Invitrogen) at a bead to cell ratio of 2:1. Tregs cells received rapamycin (50ng / ml) at the same time as activation and on day 4. Finally, on days 0, 2, 4, IL-2 (1000 units / ml) was added.
[0620] CAR construct
[0621] The anti-mouse IL23R CAR construct used in this experiment (for example, having the nucleic acid sequence of SEQ ID NO:77 and the amino acid sequence of SEQ ID NO:78) contained a human CD8 leader sequence (CD8), a crossover for human / mouse IL23R (αIL23R). Reactive scFv, hinge ...
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